Germline, loss-of-function mutations in the human retinoblastoma gene confer hereditary susceptibility to a variety of cancers, most notably retinoblastoma. Since the last competitive review, research funded by this grant has been successful in cloning this tumor suppressor gene. The isolation of this gene has allowed the applicants to pursue a number of investigations of the gene, including its relationship to retinoblastoma and other human cancers and its value in the genetic diagnosis of retinoblastoma. In this competing continuation application, the applicants propose to continue their studies of this locus. In particular, the applicants and others have documented the existence of patients who are mosaic for mutations of the retinoblastoma gene; they now wish to ascertain the frequency of such mosaicism. Another study involves a more precise measurement of the percentage of new germline mutations that occur on the paternally derived copy of the retinoblastoma gene, as opposed to the maternally derived copy. The applicants will explore the incidence of and significance of methylation of the 5' end of the retinoblastoma gene. Experiments aimed at identifying the factors important in the regulation of transcription of the retinoblastoma gene will be carried out. Finally, the applicants propose to demarcate functional domains of the protein encoded by the retinoblastoma gene by searching for regions of the primary structure that are conserved through evolution. Many of these studies have direct relevance to the clinical care of patients with retinoblastoma and their families.